Development and application of a triplex fluorescence quantitative PCR detection method for major viral pathogens in Micropterus salmoides and Siniperca chuatsi

Objective This study aimed to develop a triplex fluorescence quantitative PCR method that was able to simultaneously detect ranavirus （RanaV）， infectious spleen and kidney necrosis virus （ISKNV） and Perciformes fish Rhabdoviridae （PFRV）， with a goal of providing a convenient and efficient detection method for quarantine， monitoring， and clinical diagnosis of RanaV， ISKNV， and PFRV.Method Specific primers and TaqMan-MGB probes were designed based on the conserved sequences of the RanaV-MCP gene， ISKNV-MCP gene and PFRV-G gene. Target gene fragments were cloned into the pGM-T vector to construct recombinant plasmids of pGM-T-MCP^RanaV， pGM-T-MCP^ISKNV， and pGM-T-G^PFRV. pGM-T-MCP^RanaVand pGM-T-MCP^ISKNV served as the standard DNA for RanaV and ISKNV， respectively， while pGM-T-G^PFRV was digested with SalⅠ and then subjected to in vitro transcription to obtain the PFRV standard RNA. With the obtained standards as templates， reaction conditions were optimized and the standard curves were established to conduct the sensitivity， specificity， reproducibility tests and experiments of clinical application.Result The optimized reaction system of the triplex fluorescence quantitative PCR 30.0 μL： 2×Fast One Step Probe RT-qPCR Mix 15.0 μL， primer RanaV-qF/RanaV-qR（10 μmol/L） 0.6 μL， primer ISKNV-qF/ISKNV-qR（10 μmol/L） 0.6 μL， primer PFRV-qF/PFRV-qR（10 μmol/L） 0.6 μL， probe RanaV-qP（5 μmol/L） 0.9 μL， probe ISKNV-qP（5 μmol/L）0.9 μL， probe PFRV-qP（5 μmol/L）1.2 μL， nucleic acid templates 3.0 μL， sterile DEPC-treated water added to adjust the final volume to 30.0 μL. The triplex fluorescence quantitative PCR method demonstrated high amplification efficiency for RanaV， ISKNV and PFRV standards （Eff.>95.0%）， exhibited a strong linear relationship of standard curves（RSq≥0.999）， enabling simultaneous quantitative analysis of RanaV， ISKNV and PFRV. This method showed high detection sensitivities for 10 copies/reaction for RanaV， ISKNV and PFRV standards and 10 copies/mg for tissue samples of Micropterus salmoides and Siniperca chuats. No cross reaction was found with common fish pathogens such as grass carp reovirus （GCRV）， tilapia lake virus （TiLV）， and spring viremia of carp virus （SVCV）. The C_t variation coefficients of intra-group and inter-group for the detection of RanaV， ISKNV and PFRV were lower than 2.00%. The triplex fluorescence quantitative PCR method was applied to detect samples collected from aquaculture farms （95 pieces of Micropterus salmoides and 68 pieces of Siniperca chuats）， and the RanaV， ISKNV， and PFRV positive rates were 19.0%， 14.7%， and 4.3%， respectively.Conclusion The triplex fluorescence quantitative PCR method based on probe TaqMan-MGB could simultaneously detect RanaV， ISKNV， PFRV. It features high sensitivity， strong specificity， accuracy， reliability， simplicity， and high efficiency， providing a more convenient and effective tool for fry quarantine， epidemic monitoring， and clinical diagnosis of Micropterus salmoides and Siniperca chuats. In aquaculture in Giangxi， RanaV， ISKNV， PFRV infections are observed， as Micropterus salmoides infected by RanaV and Siniperca chuats by ISKNV， and cases of mixed infection and latent infection without clinical symptom are reported.